Heavy metals bioaccumulation in free-ranging South American rattlesnakes (Crotalus durissus) in Southeastern Brazil.

Anthropogenic activities are the main sources of soil, air, and water pollution by metals, including cadmium (Cd), lead (Pb), chromium (Cr), the metalloid arsenic (As), magnesium (Mg), zinc (Zn), and copper (Cu). The goal of this study was to assess the presence and concentration of toxic (As, Cd, Pb, and Cr) and essential metals (Mg, Zn, and Cu) in the liver and kidneys from 96 free-ranging rattlesnakes (Crotalus durissus) from Minas Gerais (Brazil). Bioaccumulation of Cd and Pb were significantly higher in males and heavier rattlesnakes (those with body weight above the average of the study population). Average ± standard deviations of Cd, Pb, Cr, Cu, Mg, Zn, and As in the general population (n = 96) were 3.19 ± 2.52; 5.98 ± 8.49; 0.66 ± 1.97; 3.27 ± 2.85; 776.14 ± 2982.92; 27.44 ± 29.55; and 0.32 ± 1.46; respectively. Bioaccumulation of some metals correlated positively with changes in hematologic and serum biochemical parameters. Results of this study were contrasted with previous studies assessing metal bioaccumulation in other species of terrestrial or aquatic snakes. Considering their position in the food chain and the broad range of bioaccumulation of both toxic and essential metals observed in this study, rattlesnakes may function as highly relevant biological sentinels for environmental pollution.

BoagamaPLI: structural and functional characterization of the gamma phospholipase A2 plasma inhibitor from the non-venomous Brazilian snake Boa constrictor

Plasma in several organisms has components that promote resistance to envenomation by inhibiting specific proteins from snake venoms, such as phospholipases A2 (PLA2s). The major hypothesis for inhibitor’s presence would be the protection against self-envenomation in venomous snakes, but the occurrence of inhibitors in non-venomous snakes and other animals has opened new perspectives for this molecule. Thus, this study showed for the first time the structural and functional characterization of the PLA2 inhibitor from the Boa constrictor serum (BoagamaPLI), a non-venomous snake that dwells extensively the Brazilianterritory. Therefore, the inhibitor was isolated from B. constrictor serum, with 0.63% of recovery. SDS-PAGE showed a band at ~25 kDa under reducing conditions and ~20 kDa under non-reducing conditions. Chromatographic analyses showed the presence of oligomers formed by BoagamaPLI. Primary structure of BoagamaPLI suggested an estimated molecular mass of 22 kDa. When BoagamaPLI was incubated with Asp-49 and Lys-49 PLA2 there was no severe change in its dichroism spectrum, suggesting a non-covalent interaction. The enzymatic assay showed a dose-dependent inhibition, up to 48.2%, when BoagamaPLI was incubated with Asp-49 PLA2, since Lys-49 PLA2 has a lack of enzymatic activity. The edematogenic and myotoxic effects of PLA2s were also inhibited by BoagamaPLI. In summary, the present work provides new insights into inhibitors from non-venomous snakes, which possess PLIs in their plasma, although the contact with venom is unlikely.

BoagamaPLI: structural and functional characterization of the gamma phospholipase A2 plasma inhibitor from the non-venomous Brazilian snake Boa constrictor

Plasma in several organisms has components that promote resistance to envenomation by inhibiting specific proteins from snake venoms, such as phospholipases A2 (PLA2s). The major hypothesis for inhibitor’s presence would be the protection against self-envenomation in venomous snakes, but the occurrence of inhibitors in non-venomous snakes and other animals has opened new perspectives for this molecule. Thus, this study showed for the first time the structural and functional characterization of the PLA2 inhibitor from the Boa constrictor serum (BoagamaPLI), a non-venomous snake that dwells extensively the Brazilianterritory. Therefore, the inhibitor was isolated from B. constrictor serum, with 0.63% of recovery. SDS-PAGE showed a band at ~25 kDa under reducing conditions and ~20 kDa under non-reducing conditions. Chromatographic analyses showed the presence of oligomers formed by BoagamaPLI. Primary structure of BoagamaPLI suggested an estimated molecular mass of 22 kDa. When BoagamaPLI was incubated with Asp-49 and Lys-49 PLA2 there was no severe change in its dichroism spectrum, suggesting a non-covalent interaction. The enzymatic assay showed a dose-dependent inhibition, up to 48.2%, when BoagamaPLI was incubated with Asp-49 PLA2, since Lys-49 PLA2 has a lack of enzymatic activity. The edematogenic and myotoxic effects of PLA2s were also inhibited by BoagamaPLI. In summary, the present work provides new insights into inhibitors from non-venomous snakes, which possess PLIs in their plasma, although the contact with venom is unlikely.

Antimicrobial photodynamic therapy for infectious stomatitis in snakes: Clinical views and microbiological findings.

BACKGROUND: Antimicrobial photodynamic therapy (APDT) has been broadly investigated as an alternative to treat localized infections, without leading to the selection of resistant microorganisms. Infectious stomatitis is a multifactorial disease frequently reported in captive snakes characterized by infection of the oral mucosa and surrounding tissues. In this study, we investigated methylene blue (MB)-mediated APDT to treat infectious stomatitis in snakes and verified the resistance phenotype and genotype before and after APDT. METHODS: Three Boid snakes presented petechiae, edema and caseous material in their oral cavities. MB (0.01%) was applied on the lesions and after 5min they were irradiated using a red laser (λ=660nm), fluence of 280J/cm2, 8J and 80s per point, 100mW, spot size 0.028cm2 and fluence rate of 3.5W/cm2. APDT was repeated once a week during 3 months. Samples of the lesions were collected to identify bacteria and antibiotic resistance profiles. To analyze the clonality of bacterial isolates before and after APDT, isolates were subjected to ERIC PCR analysis. RESULTS: Snakes presented clinical improvement such as reduction of inflammatory signs and caseous material. Pseudomonas aeruginosa and Escherichia coli were present in all snakes; Klebsiella pneumoniae and Morganella morganii were also identified in some animals. We also observed that the oral microbiota was completely replaced following APDT. However, K. pneumoniae isolates before and after APDT were a single clone with 100% of genetic similarity that lost resistance phenotype for seven antibiotics of four classes. CONCLUSIONS: These results show that APDT can be used to treat infectious stomatitis in snakes.

Histology of the gastrointestinal tract from Bothrops jararaca and Crotalus durissus / Aspectos histológicos do trato gastrointestinal de Bothrops jararaca e Crotalus durissus

Recent studies have discussed the importance of snakes, going beyond the context of Public Health (snakebites). Promising research demonstrates the utility of snake venom in several fields, including oncology, diagnosis, anticoagulant therapy, and pain treatment. Nevertheless, there are still gaps in the basic knowledge on these animals, specifically regarding the histological characterization of the gastrointestinal tract, which ultimately hinder comparative histology and pathology studies. In this context the present study intends to contribute with the advance of current knowledge on snake histology by describing and analyzing histological samples of the gastrointestinal tracts of Bothrops jararaca and Crotalus durissus. Samples were collected from 12 individuals (six from each species), up to 6 months of age and equally distributed between males and females. Histological slides were prepared from the organs collected and were stained with hematoxylin and eosin, periodic acid-Schiff and alcian blue. The slides were photographed with a high-resolution camera to create a portfolio representative of the histology of the gastrointestinal systems of these animals.(AU)

Na atualidade, a importância das serpentes extravasa a conotação em saúde pública (acidentes ofídicos), e pesquisas promissoras têm demonstrado a utilidade dos venenos das serpentes em diversas áreas como oncologia, diagnóstico, terapia anticoagulante e tratamento da dor. Ainda assim, existem lacunas no conhecimento básico sobre esses animais, tais como a caracterização histológica do seu sistema gastrointestinal, que se tornam obstáculos para estudos nos campos da histologia e patologia comparadas. Nesse contexto, o presente trabalho pretende contribuir com o avanço do conhecimento sobre histologia de serpentes a partir da descrição e análise histológica de amostras do trato gastrointestinal de exemplares das espécies Bothrops jararaca e Crotalus durissus. Para tanto, foram coletadas amostras de 12 indivíduos com até seis meses de idade, seis por espécie (distribuídos igualmente entre machos e fêmeas). A partir dos órgãos colhidos, lâminas histológicas foram preparadas e coradas pela técnica de hematoxilina e eosina, bem como PAS com Alcian Blue. As lâminas foram fotografadas por câmera de alta resolução, resultando em um portfólio representativo da histologia do sistema gastrointestinal desses animais.(AU)

Microcomputed tomographic, morphometric, and histopathologic assessment of congenital bone malformations in two neotropical viperids

Congenital malformations have been reported in all classes of vertebrates and may be a determinant of life span and survival. In reptiles, the incidence of congenital malformations can be associated with genetic and environmental causes, including pollution. The characterization of pathological processes involved in the development of congenital malformations of bone in snakes is rare in the literature, but is of great relevance in the field of reptile conservation and environmental health. We describe congenital bone lesions in 50 newborn jararaca (Bothrops jararaca) and 26 South American rattlesnakes (Crotalus durissus terrificus) born from wild-caught pregnant females in Southeastern Brazil. Lesions were evaluated by morphometric quantitative analysis, x-ray microtomography, and histopathologic descriptive analysis. Morphometric analysis showed that jararaca presented more severe axial lesions (kyphosis, scoliosis, and kyphoscoliosis) than rattlesnakes. Female rattlesnakes presented more severe axial lesions than did males. In rattlesnakes, spinal deformities were more frequently diagnosed in the caudal segment of the body. We present x-ray microtomographic assessments and images of malformed snakes (n=9) and characterized novel malformations, such as the agenesis of frontal, parietal, and supraoccipital bones in a jararaca specimen. Histopathologic findings included vertebral body fusion, myositis, coagulation necrosis, and disorganization of periaxial muscle fibers. The new methods and results presented in this study will be useful and informative for future research in pathology, teratology, embryology, and ecotoxicology in snakes.

Antimicrobial photodynamic therapy for infectious stomatitis in snakes: clinical views and microbiological findings

Background Antimicrobial photodynamic therapy (APDT) has been broadly investigated as an alternative to treat localized infections, without leading to the selection of resistant microorganisms. Infectious stomatitis is a multifactorial disease frequently reported in captive snakes characterized by infection of the oral mucosa and surrounding tissues. In this study, we investigated methylene blue (MB)-mediated APDT to treat infectious stomatitis in snakes and verified the resistance phenotype and genotype before and after APDT. Methods Three Boid snakes presented petechiae, edema and caseous material in their oral cavities. MB (0.01%) was applied on the lesions and after 5 min they were irradiated using a red laser (Lambda = 660 nm), fluence of 280 J/cm2, 8 J and 80 s per point, 100 mW, spot size 0.028 cm2 and fluence rate of 3.5 W/cm2. APDT was repeated once a week during 3 months. Samples of the lesions were collected to identify bacteria and antibiotic resistance profiles. To analyze the clonality of bacterial isolates before and after APDT, isolates were subjected to ERIC PCR analysis. Results Snakes presented clinical improvement such as reduction of inflammatory signs and caseous material. Pseudomonas aeruginosa and Escherichia coli were present in all snakes; Klebsiella pneumoniae and Morganella morganii were also identified in some animals. We also observed that the oral microbiota was completely replaced following APDT. However, K. pneumoniae isolates before and after APDT were a single clone with 100% of genetic similarity that lost resistance phenotype for seven antibiotics of four classes. Conclusions These results show that APDT can be used to treat infectious stomatitis in snakes.

Functional characterization of neotropical snakes peripheral blood leukocytes subsets: linking flow cytometry cell features, microscopy images and serum corticosterone levels

Reptiles are the unique ectothermic amniotes, providing the key link between ectothermic anamniotes fish and amphibians, and endothermic birds and mammals; becoming an important group to study with the aim of providing significant knowledge into the evolutionary history of vertebrate immunity. Classification systems for reptiles' leukocytes have been described by their appearance rather than function, being still inconsistent. With the advent of modern techniques and the establishment of analytical protocols for snakes' blood by flow cytometry, we bring a qualitative and quantitative assessment of innate activities presented by snakes’ peripheral blood leukocytes, thereby linking flow cytometric features with fluorescent and light microscopy images. Moreover, since corticosterone is an important immunomodulator in reptiles, hormone levels of all blood samples were measured. We provide novel and additional information which should contribute to better understanding of the development of the immune system of reptiles and vertebrates.

Histological analysis of the eyeball of Neotropical birds of prey Caracara plancus, Falco sparverius, Rupornis magnirostris, Megascops choliba and Athene cunicularia / Análise histológica do bulbo ocular de aves de rapina neotropicais: Caracara plancus, Falco sparverius, Rupornis magnirostris, Megascops choliba e Athene cunicularia

The growing use of ophthalmic examination as a screening tool in birds intended for reintroduction into natural environments over the last few years has given renewed significance to avian ophthalmology in the context of free-ranging and captive bird conservation. The eye plays a vital role in prey detection and capture by birds of prey. The remarkable eyesight of such birds makes them interesting subjects for avian visual system anatomical and histological investigation. This study set out to describe histological features of the eyeball of ubiquitous birds of prey in Brazil (Falconiformes, Accipitriformes and Strigiformes). Twenty enucleated cadaveric eyeballs obtained from birds with natural death, Caracara plancus, Falco sparverius, Rupornis magnirostris, Megascops choliba and Athene cunicularia were used. Routinely prepared histological slides stained with hematoxylin and eosin were analyzed under light microscopy. Similarities and variations in ocular structures between the different bird species studied were highlighted in this study, with major differences concerning the lens and retina. This study highlights the importance of determining the ocular histological pattern of the species so they can be better understood. These results may well assign baseline information of the species and assist in eye histopathological diagnostics.(AU)

Nos últimos anos a oftalmologia aviária se tornou um instrumento de grande relevância para a conservação de aves de vida livre e de cativeiro, principalmente pela crescente utilização do exame oftalmológico para a triagem de indivíduos designados para reintrodução em ambientes naturais. Os olhos das aves de rapina são estruturas imprescindíveis para detectar e capturar suas presas, e a sua notável capacidade visual torna o seu sistema de visão assunto de grande interesse para o estudo anatômico e histológico. Neste contexto, no presente trabalho foi realizada a análise histológica do bulbo ocular de aves de rapina representadas por espécies pertencentes às ordens Falconiformes, Accipitriformes e Strigiformes presentes em território brasileiro. Para realização do estudo foram utilizados vinte bulbos oculares obtidos de animais mortos por causas naturais, das espécies Caracara plancus, Falco sparverius, Rupornis magnirostris, Megascops choliba e Athene cunicularia.. As lâminas histológicas foram coradas pela técnica de hematoxilina e eosina e, analisadas por microscopia óptica. Foi constatada a existência de um padrão histológico com discreta variação entre as estruturas oculares nas diferentes espécies avaliadas, com destaque para o cristalino e a retina. Este estudo salienta a importância da determinação do padrão histológico ocular das espécies, e seus resultados atribuem informações histológicas basais auxiliares na determinação de diagnósticos histopatológicos oculares.(AU)